Protein kinase A mediates inhibition of the thrombin-induced platelet shape change by nitric oxide

Abstract

The thrombin-induced platelet shape change was blocked by nitric oxide (NO), as revealed by scanning electron microscopy, light transmission, and resistiveparticle volume determination. The inhibitory effect of NO was accompanied by an increase in levels of both cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). However, the inhibition of the shape change was only mimicked by cAMP analogs (Sp-5,6-DClcBIMPS, 8-AHA-cAMP, and 8-CPT-cAMP) and not by cGMP analogs (8-Br-PETcGMP, 8-Br-cGMP, and 8-pCPT-cGMP). The effect of NO on the thrombin-induced shape change was prevented by the protein kinase A (PKA) antagonists Rp-8-BrcAMPS and Rp-cAMPS. The protein kinase G (PKG) antagonist Rp-8-CPTcGMPS strongly inhibited PKG-mediated 46-kDa VASP Ser239 phosphorylation, but did not inhibit the thrombin-induced shape change or the PKA-mediated VASP Ser157 phosphorylation. Whereas an inhibitor of cyclic nucleotide phosphodiesterase (PDE) 3A (milrinone) mimicked the effect of NO, inhibitors of PDE2 (erythro-9-(2-hydroxy-3-nonyl)adenine) and PDE5 (dipyridamole) were poorly effective. We concluded that (1) NO was a potent and reversible inhibitor of the platelet shape change, (2) the shape change was reversible, (3) the inhibitory effect of NO was mediated through activation of PKA, (4) the onset of the NO effect coincided with VASP Ser157 phosphorylation, and (5) removal of NO and platelet shape change coincided with VASP Ser157 dephosphorylation. These findings are compatible with elevation of cGMP by NO in a compartment close to PDE3A, PKA, and VASP, leading to a local increase of cAMP able to block thrombin-induced shape change. IntroductionWhen platelets are stimulated, they rapidly change shape from discoids to spheres possessing extensive pseudopodia. [1][2][3] This shape change precedes aggregation, secretion, and activation of the coagulation cascade. 1,2,4 In healthy individuals, 65% to 90% of circulating platelets are discoidal, 2 and increased numbers of spherical platelets have been associated with vascular diseases. 5 Shape change could therefore be an important step in hemostasis and thrombosis. Activation of the platelet shape change by thrombin has been extensively studied in vitro, but little is known about mechanisms inhibiting this process. Thrombin, a potent platelet agonist that induces shape change, aggregation, and secretion through many signaling systems 6 is formed in vivo, in significant amounts at the site of a vascular injury, stimulating both platelet activation and overall thrombus growth. [7][8][9][10][11][12] Regulation of platelet activation is an important physiologic function of the vascular endothelium including release of nitric oxide (NO), 13 a potent inhibitor of platelet aggregation, secretion, adhesion, and binding of fibrinogen to integrin glycoprotein IIbIIIa (GpIIbIIIa). [14][15][16][17] Conflicting results have, however, been reported about the effect of NO on shape change. [18][19][20][...

Platelet Resistance to the Antiaggregatory Cyclic Nucleotides in Central Obesity Involves Reduced Phosphorylation of Vasodilator-Stimulated Phosphoprotein

“…Because the molecular defects observed in obese individuals are involved in crucial steps of the control of platelet function (12)(13)(14)(15)(16), the present results may be relevant in explaining resistance not only to the antiaggregatory effects of the cyclic nucleotide analogs but also to other antiaggregatory mediators, which we investigated in previous studies (6,9,11 ). The reduced antiaggregatory effects of insulin, organic nitrates, and prostacyclin, which act through activation of cyclic nucleotide/protein kinase pathways (10,11 ), may be explained, at least in part, by an impaired action of the cyclic nucleotides on their specific kinases.…”

“…Most authors agree that the main effect of cyclic nucleotides is inhibitory and is exerted through activation of the corresponding cyclic nucleotide-dependent protein kinases, i.e., protein kinase G (PKG) 1 for cGMP and protein kinase A (PKA) for cAMP (12)(13)(14)(15)(16), which are involved in the regulation of basic mechanisms of platelet activation, such as agonist-induced increases of cytosolic calcium (12,(15)(16)(17), fibrinogen binding (18 ), and cytoskeleton protein contraction (19 ). Recent evidence indicates, however, that increased concentrations of platelet cGMP are associated with enhanced platelet function (20,21 ).…”

cAMP inhibits modulation of airway smooth muscle phenotype via the exchange protein activated by cAMP (Epac) and protein kinase A

“…Following serum deprivation, cells were treated with 16,16-dimethyl-PGE2, 8-pCPT-2′-O-MecAMP and/or 6-Bnz-cAMP in the absence or presence of PDGF for 4 days. In some experiments cells were pretreated for 30 min with the prostaglandin EP2 receptor antagonist AH6809 (1 mM; receptor nomenclature follows Alexander et al, 2009), H89, in a PKA-selective concentration (300 nM) or with a mixture of two PKA inhibitors (Rp-cAMPS and Rp-8-Br-cAMPS, 500 mM, each) (Jensen et al, 2004). After 4 days, cells were washed twice with HBSS and incubated with a 5% vol/vol Alamar blue in HBSS for 45 min.…”

“…The involvement of PKA was demonstrated by treatment with the PKA inhibitor H89, which partially reduced the inhibitory effect of 16,16-dimethyl-PGE2 on PDGF-induced cell proliferation (P < 0.05; Figure 1D). Because reported nonspecific effects of H89 might render interpretation of the results difficult (Davies et al, 2000), we also used a combination of two selective PKA antagonist, Rp-cAMPS and Rp-8-Br-cAMPS (Jensen et al, 2004). Combined treatment with Rp-cAMPS and Rp-8-BrcAMPS (500 mM; each) fully inhibited the effect of the PGE2 derivative (P < 0.05; Figure 1E).…”

Ticagrelor Compared with Clopidogrel Increased Adenosine and Cyclic Adenosine Monophosphate Plasma Concentration in Acute Coronary Syndrome Patients

“…Ticagrelor could increase the adenosine and cAMP plasma concentration by inhibiting the uptake through the ENT. Adenosine and cAMP inhibit platelet activation mainly via A 2A R but also via A 2B R [21,22]. The main mechanism of the weak correlation with adenosine and antiplatelet effect might be that ticagrelor had a low affinity on platelet A 2A receptors [19].…”